In the field of genetic engineering, precise controlling gene expression plays an important role in studying gene function and life processes of living organisms. Gene expression system in prokaryotic bacterium is much simpler relative to the complex gene expression system in eukaryotic cells. Take the most widely used prokaryotic bacterium E. coli for example, the first step is the transcription of DNA into RNA by RNA polymerase. The RNA polymerase of E. coli consists of five subunits; its molecular weight is about 480 Kd, it contains α, β, β′, σ four different polypeptides, there are two molecular of α polypeptide, so the holoenzyme is α2ββ′ σ. α subunit is linked with the formation of tetramer core enzyme (α2ββ′) of RNA polymerase; β subunit contains the binding site of nucleoside triphosphate; β′ subunit has the binding site of DNA template; σ is only linked with the initiation of RNA transcription and is not unrelated to the elongation of chain. Once transcription initiates, σ is released and the elongation of chain is catalyzed by tetramer core enzyme. So the function of σ is recognition of the transcriptional initiation signal and enabling binding of RNA polymerase to the promoter region. The initiation signal in DNA, also as “initiation sequence”, is termed as promoter. The promoter of E. coli consists of −10 region and −35 region, −10 region locates at 10 bp upstream of the transcriptional start point and contains the conserved sequence of six bases TATATA which is the tight binding site of RNA polymerase. Another conserved sequence of six bases TTGACA is located at 35 bp upstream of the transcriptional start point, −35 region provides the recognition signal of RNA polymerase; the promoter activity of E. coli depends on the base contents of −10 and −35 regions and space length between −10 and −35 regions. Although the core enzyme can bind to the DNA, it mainly results from the non-specific electrostatic attraction between basic protein and acidic nucleic acid, the DNA is still double helix, σ subunit can alter the affinity of RNA polymerase and DNA and significantly increase the binding constant and residence time of the enzyme and promoter. The core enzyme contacts with DNA with the help of σ subunit and forms non-specific complex, such complex is not stable and the enzyme can slide along with the DNA chain. The holoenzyme rapidly recognizes the promoter with the help of 6 subunit and binds to it to form relaxed closed promoter complex. The RNA polymerase binds to DNA surface and the recognition is located at the −35 region of promoter. Then the conformation of DNA changes to form the open promoter complex, at this time, the enzyme binds tightly to the promoter, untwists the double strands of DNA at −10 region and recognizes the template strand. It is easy to untwist the DNA strands due to the region containing rich A-T base. Once the formation of open complex, DNA continues to be untwisted and the enzyme moves to the transcription start point. Bacillus is another widely used prokaryotic bacteria and is gram-positive bacterium. Somewhat differently, Bacillus contains many kinds of RNA polymerases and σ which recognize different promoter sequences.
The gene expression systems of prokaryotic bacteria can be divided into two types, the first is constitutive expression which enables the independent continuous expression of target gene without induction. The other is inducible gene expression system which can be divided into small chemical induced gene expression system and physical methods induced gene expression system according to the inducers. For the small chemical induced gene expression system, IPTG is the mostly used inducer. IPTG is the analogue of lactose and has extremely strong induction ability, it is very stable and cannot be metabolized by bacteria. The inducer of current most widely used expression vectors containing T7 promoter, lac promoter, Tac promoter and grac promoter is IPTG. Expression systems using arabinose and tryptophan as the inducers have been used more and more, arabinose and tryptophan have the advantages of no toxicity and tight regulation. The discovery of Mn2+, Fe2+, Cu+ et al metal ions sensing proteins attracts peoples' eyes to use the metal ions binding proteins to induce protein expression. Using the changes of temperature to induce gene expression is widely used in physical methods induced gene expression system, such as the temperature sensitive mutant of lad which repress the promoter activity at 30° C. and loose its activity and cannot repress the promoter activity at 42° C. Ultraviolet (UV)-regulated “cage” (Caged) technology [Keyes, W M and A A Mills, Trends Biotechnology, 2003, 21 (2): 53-55]1 is another widely used physical methods inducible gene expression system.
Although many of those methods have been widely used, there exist some potential problems: (1) some inducers have great toxicity and are expensive (IPTG), it is not suitable to expression recombinant proteins for gene therapy; (2) in metal ions inducible gene expression systems, the recognition of metal ion sensing proteins to metal ions lacks specificity, different metal ions of the same family or the same period can be recognized by the same sensing protein to activate the transcription, so the transcription can be interfered many metal ions in the internal environment of prokaryotic bacteria cells. Additionally, the low valence metal ions can be oxidized by the oxidizing environment of prokaryotic bacteria cells, resulting in interference of transcriptional activation by the metal ions that need strict oxidizing environment; (3) In the temperature inducible gene expression systems, the increase of external temperature can activate the heat shock proteins of E. coli to affect the stability of products, some proteins are difficult to fold correctly, the UV-induced cage technology may cause irreversible damage to cells; (4) the most importantly, chemical inducers only can temporally regulate gene expression, but cannot spatially regulate the gene expression in specific cells and tissues.
Nevertheless, light is easy to be spatiotemporally manipulated, has no toxicity to cells and is easy to obtain. In recent years, light-regulated proteins (also known as photosensitive protein) was found in the biological clock systems of some organisms, its functions can be significantly affected by light illumination. We aimed to engineer the natural existing transcription factor to obtain artificial light-sensitive transcription factor based on molecular design, and in turn construct light-switchable gene expression system in prokaryotic bacterium. However, studies on light-regulated transcription factors have been rarely reported, there are only two systems. Anselm Levskaya et al. reported a light regulated protein expression system based on the phytochrome Cph1 and EnvZ/OmpR two-component of E. coli in 2005 [Levskaya, A. et al, Nature, 2005. 438(7067): p. 441-2.]2. In the dark conditions, the light-switchable transcription factor autophosphorylated and bond to OmpR dependent ompC promoter, and then initiated the transcription and expression of target gene. Upon red light illumination, the autophosphorylation of light-switchable transcription factor was inhibited and could not bind to ompC promoter, so the transcription and expression of target gene could not be activated. In the following years, this light regulated protein expression system was modified by the same group to obtain multi-color co-regulated protein expression systems [Tabor, J. J. et al., J Mol Biol, 2011. 405(2): p. 315-24, Tabor, J. J. et al., Cell, 2009. 137(7): p. 1272-81.]3, 4. Keith Moffat group developed another novel light-switchable transcription factor YF1 which was based on blue light sensitive protein YtvA from Bacillus subtilis and FixL protein from Bradyrhizobium japonicum [Moglich, A. et al., J Mol Biol, 2009. 385(5): p. 1433-44, Ohlendorf, R. et al., J Mol Biol, 2012. 416, 534-542.]5, 6. Gene expression from the light regulated gene expression system based on YF1 was repressed upon blue light illumination, and high gene expression occurred without blue light. However, both of the two systems had marked limitations. In addition to the light-switchable transcription factor and reporter system, the first system is very complex, it is necessary to introduce ho1 and pcyA genes into cells to obtain the required phycocyanobilin from haem, which significantly increases the work of system construction. The second system has high leak expression even upon blue light illumination and has only dozens of the induction ratio, so it is difficult to precisely control gene expression. The above described disadvantages limit the use of these two systems in prokaryotic bacterium. Until now, except for the used photosensitive proteins Cph1 and YtvA, there are some other known photosensitive proteins: the photosensitive proteins using flavin as the chromophore (also called flavin-containing protein family blue light receptor), which can be divided into three groups: first is photoreceptors with light-oxygen-voltage (LOV) domain, such as phytochrome; the second is photolyase-like cryptochromes; the third is blue light using FAD (BLUF) family that is found in recent years.
Phytochrome is the most common photoreceptor containing LOV domain, such as phototropin 1, white collar-1 (WC-1), white collar-2 (WC-2), photoactive yellow protein (PYP), Phy3, VVD, etc. Phytochrome is usually a membrane-coupled kinase which can autophosphorylate and alters its activity to regulate specific physiological processes upon blue light exposure. Most phytochromes have Serine/Threonine kinase domain at the C-terminal and two LOV domains with flavin at the N-terminal. With the illumination of blue light, the LOV domain and flavin bind covalently to form a cysteinyl-flavin adduct which can cause the conformation change of flavin-binding pocket and then enable the kinase domain at the C-terminal to alter the kinase activity. This process is reversible. The most successful light regulated gene expression system in eukaryotic cells is based on photosensitive protein VIVID. Yang's group [Wang, X. et al., Nat Methods, 2012. p. 266-269.]7 developed a eukaryotic light-switchable gene expression system based on the formation of homodimer of blue light sensitive protein VIVID from Neurospora crassa after blue light illumination. In this system, light-switchable transcription factor consists of three or four polypeptides, the ability of dimerization of the recombinant light-switchable transcription factor changed after blue light illumination, the dimerized transcription factor bond to the reaction element of the target transcription unit nucleotide sequence, to regulate (initiate/repress) the transcription and expression of the target gene via the synergistic effect on the promoter in the target transcription unit by the transcriptional activation/repression domain of the third polypeptide in this fusion protein and other transcriptional co-factors derived from the recruitment host cells. This system is considered as the best gene expression system of eukaryotic cells due to the following advantages: simplicity, fast induction kinetics, high induction ratio, good reversibility and high spatiotemporal resolution. However, it is a pity that the transcription and translation mechanism of prokaryotic bacteria differs from that of eukaryotic cells, so this system cannot be used in prokaryotic bacteria. Masayuki Yazaw et al. [Yazawa, M. et al., Nat Biotechnol, 2009. 27(10): p. 941-5] also developed a eukaryotic light regulated gene expression system based on the interaction of FKF1 (flavin-binding, kelch repeat, f box 1) and GI (GIGANTEA) from Arabidopsis thaliana upon blue light illumination, but its application was limited for the low induction ratio and complexity of the system.
Cryptochromes from Arabidopsis thaliana are the first separated blue light photosensitive plant proteins, of which some have been well studied, such as cryptochrome1 (CRY1), cryptochrome 2 (CRY2), phytochrome A (phyA) and phytochrome B (phyB), their functions were regulated by the light of circadian rhythm to control growth and movement of plants. The amino acid sequences and fluorophore of cryptochromes are the similar to photofragmentation proteins, the molecular weight of most cryptochromes is about 70 kD-80 kD, it contains conservative PHR domain (relevant to photofragmentation enzyne) at the N terminal and unknown domain with great differences in length at the C terminal, the PHR domain can non-covalently bind to flavin. Based on the interaction of Arabidopsis CRY2 and CIB1 (CRY-interacting bHLH1) protein upon blue light illumination, people developed a light regulated gene expression system in eukaryotic cells [Kennedy, M. J. et al, Nat Methods, 2010. 7(12): p. 973-5.]8.
Difference between blue light photoreceptor proteins with BLUF domain and photoreceptor proteins with LOV domain is that no adduct is generated between BLUF and flavin after light irradiation, but it will lead to 10 nm red-shift absorbance due to the comformation change of chromophore. The most well studied BLUF domain containing photoreceptor is AppA, which is a repressor of anti-transcription from Rhodobacter sphaeroides. AppA and transcription factor PpsR combine to form AppA-PpsR2 complex and enable PpsR not to bind with DNA in darkness; bright blue light irradiation may enable AppA to dissociate from the complex, and the released PpsR forms a tetramer and bind to a specific DNA sequence to repress the gene transcription [Pandey, R. et al, FEBS J, 2012.]9.
Haifeng Ye et al. [Ye, H. et al, Science, 2011. 332(6037): p. 1565-8.]10 developed a blue light activated light induced gene expression system of eukaryotic cells based on melanopsin and intracellular signaling. Melanopsin is a photosensitive protein of certain retinal cells. Upon blue light illumination, melanopsin rapidly triggers the influx of Ca2+ into cells, after a series of cascade, calmodulin activates the serine/threonine phosphatase calcineurin, which dephosphorylates the transcription factor NFAT, the dephosphorylated transcription factor NFAT enters into nucleus and bind to the NFAT-dependent promoter to activate transcription and translation of target genes. The drawback of this system is that it is involved in intracellular signaling, resulting in poor stability and interrupting normal life activities by affecting cell signaling.
In relative to eukaryotic cells, prokaryotic bacteria have advantages of fast proliferation, low costs and high expression of foreign proteins (even can reach 90% of the total proteins), enabling it more suitable for large scale production of interested proteins. As described above, the most widely used gene expression systems today utilize chemical substances as the inducers, which have reasonable desirable induction performance, low leakage expression and high expression levels. However, many of gene expression systems have side-effect and potential toxicity due to their pleiotropic effect. Besides, the chemical inducers cannot precisely control gene expression at high spatial resolution. Up to now, there are only a few of gene expression systems controlled by physical methods, raise of temperature results in side-effect. Few photosensitive protein based gene expression systems have been developed, but the complexity and low induction ratio may limit their wide application.
In summary, it is considered that a more excellent gene expression system of prokaryotic bacteria can be created to overcome the shortcomings of previous studies and it can be widely used in biomedical researches. After painstaking studies, the inventers have created a novel light-switchable gene expression system in prokaryotic bacterium. It has an excellent capacity to control the gene expression and it can spatiotemporally regulate the gene expression.
Accordingly, the first object of the invention is to provide a novel light-switchable gene expression system of prokaryotic bacterium.
The second object of the invention is to provide a method of the regulation of gene expression by using said light-switchable gene expression system in prokaryotic bacterium.
The third object of the invention is to provide a prokaryotic expression vector containing said light-switchable gene expression system.
The fourth object of the invention is to provide a method of the regulation of life processes (such as bacteria mobility, lysis) of prokaryotic bacterium.
The fifth object of the invention is to provide a kit comprising a prokaryotic expression vector containing said light controllable gene expression system or a prokaryotic bacterium strain containing a light-switchable transcription factor in its genome.